Solid fat nanoemulsions as vaccine delivery vehicles

ABSTRACT

The present invention provides pharmaceutical vaccine compositions that are nanoemulsions of particles having a lipid core which is in a solid or liquid crystalline phase at 25° C., and which is surrounded by at least one phospholipid bilayer for the parenteral, oral, intranasal, rectal, vaginal or topical delivery of both hydrophilic and lipophilic immunogens. The particles have a mean diameter in the range of 10 to 250 nm and the immunogen is incorporated therein, either intrinsically prior to the homogenization process or extrinsically thereafter.

CROSS-REFERENCE TO RELATED APPLICATION

The present application 371 of PCT/US94/05589 filed May 18, 1994,published as WO94/26255 Nov. 24, 1994, is a continuation-in-part of U.S.patent/ application Ser. No. 08/063,613, filed May 18, 1993 now U.S. PatNo. 5,576,016.

FIELD OF THE INVENTION

The present invention concerns methods and compositions for delivery ofvaccines by parenteral and other routes of administration. Moreparticularly, it concerns stable lipid-in-water nanoemulsions oremulsomes containing small lipid particles which are useful as deliveryvehicles for both hydrophilic and lipophilic immunogens enhancing theirimmunogenicity and improving their immune response.

BACKGROUND OF THE INVENTION

The body's immune system recognizes pathogens as foreign and is thoughtto produce antibodies to them by two main pathways. In one pathway,antigens on the pathogen surface presumably bind to receptor moleculeson the white blood cells known as B cells, causing them to become plasmacells, which proliferate and secrete antibodies specific for thepathogen. In the second pathway, circulating macrophages bind to thepathogens, endocytose them, and display processed antigens on theirsurface.

T cells then bind to the expressed antigen and by way of several complexsteps this binding ultimately results in further plasma cellproliferation and increased antibody production.

In the past, the risks of whole-pathogen vaccines and limited suppliesof useful antigens posed barriers to development of practical vaccines.Today, the tremendous advances of genetic engineering and the ability toobtain many synthetic recombinant protein antigens derived fromparasites, viruses, and bacteria has revolutionized the development ofnew generation vaccines.

Although the new, small synthetic antigens offer advantages in theselection of antigenic epitopes and safety, a general drawback of smallantigens is poor immunogenicity.

Unfortunately, the body's immune system does not respond strongly tosmall peptides. In particular, macrophages do not readily ingest andprocess the small antigens resulting in low antibody titers and the needfor repeated immunizations. This lack of immunogenicity has created anacute need to identify pharmaceutically acceptable delivery systems forthese new antigens.

Several reports describing the improvement of immune response achievedby the association of antigens with lipid carriers such as liposomes ormicroparticles like polymeric biodegradable microcapsules have beenpublished (C. R. Alving, Liposomes as Carriers of Vaccines, in"Liposomes: From Biophysics to Therapeutics", M. J. Ostro, ed., Ch. 6,Marcel Dekker Inc., New York, 1987, pp. 195-218; J. H. Eldridge et al,Molec. Immunol., 28, 287, 1991). The ability of these delivery systemsto enhance immunogenicity was related to the physicochemicalcharacteristics of the particles.

When antigens are incorporated in lipid carriers by encapsulation orentrapment, or embedded in their surface, they show enhanced ability toevoke a strong immune response. Vaccines formulated in lipid carriersprobably enhance antibody production by increasing activity along bothpathways of stimulation of immune system described above. When multipleantigens attached to a lipid carrier bind to multiple receptors on a Bcell, the resulting plasma cell probably proliferates faster than itdoes when it encounters a solitary antigen. Similarly, whereas amacrophage is unlikely to phagocytase a small antigen efficiently, itwill readily digest a lipid carrier particle containing the antigen.When phagocytosis takes place, antigens ooupled to the surface of thelipid carrier or encapsulated within the particle are ingested andpossibly displayed as processed antigen on the macrophage surface. Suchantigen presentation could result in T cell activation and additionalplasma cell proliferation and increased antibody production.

Most vaccine adjuvants are also surface-active, or have a specialsurface interface. Surface-active agents concentrate at the surfaceformed by the interface of water and non-polar substances such as lipidor lipid membrane. Most adjuvants are also water-insoluble surfactants,so lipoidal vehicles are necessary for proper delivery of the antigen.

The use of liposomes as drug delivery systems has been known for sometime, and comprehensive review articles on their properties and clinicalapplications are available; see, e.g., Barenholz and Amselem, in"Liposome Technology", 2nd ed., G. Gregoriadis, ed., CRC Press, 1992;Lichtenberg and Barenholz, in Methods for Biochemical Analysis, 33, D.Glick, ed., 1988. A liposome is defined as a structure consisting of oneor more concentric lipid bilayers separated by water or aqueous buffercompartments. These hollow structures, which have an internal aqueouscompartment, can be prepared with diameters ranging from 20 nm to 10 μm.They are classified according to their final size and preparation methodas: SUV, small unilamellar vesicles (0.5-50 nm); LUV, large unilamellarvesicles (100 nm); REV, reverse phase evaporation vesicles (0.5 μm); andMLV, large multilamellar vesicles (2-10 μm).

An extensive literature exists on immunologic characteristics ofliposomes, and numerous reviews on their potential as vaccine carriershave been published especially by C. R. Alving and co-workers whodeveloped the first injectable liposomal vaccine for human useadministered to 30 volunteers in a Phase I study (Fries et al, Proc.Natl. Acad. Sci. USA, 89, pp. 358-362, 1992).

Emulsions are defined as heterogeneous systems of one liquid dispersedin another in the form of droplets usually exceeding 1 μm in diameter.The two liquids are immiscible and chemically unreactive or slowlyreactive. An emulsion is a thermodynamically unstable dispersed system.Instability is a result of the system's tendency to reduce its freeenergy by separating the dispersed droplets into two liquid phases.Instability of an emulsion during storage is evidenced by creaming,flocculation (reversible aggregation), and/or coalescence (irreversibleaggregation).

The use of parenteral emulsions as drug delivery systems is stillcomparatively rare because of the necessity of achieving stablemicrodroplets of less than 1 μm to prevent formation of emboli in theblood vessels. In order to increase the stability and useful lifetime ofthe emulsion, the dispersed lipid droplets must be coated or treatedwith surfactants or "stabilizers", which lower the free energy at theinterface and decrease the tendency of droplets to coalesce. However,due to their detergent characteristics, most of them are hemolyticagents which act as membrane solubilizers, producing deleterious sideeffects upon injection into the body. Formulation options are severelyrestricted by the very limited selection of stabilizers and surfactantsapproved and safe for parenteral injection.

SUMMARY OF THE INVENTION

The present invention provides pharmaceutical compositions comprisingnanoemulsions of particles comprising a lipid core composed of a lipidwhich is in a solid or liquid crystalline phase at at least 25° C.,stabilized by at least one phospholipid envelope, for the parenteral,oral, ocular, rectal, vaginal, intranasal, or topical delivery of bothfat-soluble and water-soluble immunogens. The new entity is aparticulate vehicle which is denoted herein as a solid fat nanoemulsionor "emulsome." These compositions have features which are intermediatebetween liposomes and oil-in-water emulsions. Emulsome particles containa hydrophobic core, as in standard oil-in-water emulsions, butsurrounded and stabilized by one or more bilayers or envelopes ofphospholipid molecules, as in liposomes (FIGS. 1A, 1B and 1C).

A key feature of these particles is that the core is composed of a lipidwhich in bulk form is in a solid or liquid crystalline phase, ratherthan an oil in a fluid phase. Lipid compositions of the core arecharacterized as being in the solid or liquid crystal phase at at least25° C. when measured in bulk form.

Emulsomes, having the characteristics of both liposomes and emulsions,provide the advantages of high loading of hydrophobic bioactivecompounds in the internal solid lipid core and the ability toencapsulate water-soluble antigens in the aqueous compartments ofsurrounding phospholipid layers.

The present pharmaceutically stable solid fat nanoemulsions or emulsomesmay be formulated in the absence of any ionic or non-ionic nonnaturalsynthetic surfactants or cosurfactants such as polyoxamers,deoxycholate, polysorbates, tyloxapol, or emulphor. They are stabilizedby the combination of relatively high lecithin content and the use ofsolid lipid compositions as the core.

The particle size distribution of emulsomes, based on differentialweight percents, is in the range of 10-250 nm, making them suitable forparenteral administration.

The emulsome technology represents a new type of lipid-basedencapsulation technology that has potential usefulness as carriers ofvaccines and adjuvants enhancing the immunogenicity of antigensincorporated intrinsically or extrinsically into the particles.

Emulsome-vaccine formulations containing antigens can provide thesurface interphase necessary for proper orientation of the adjuvantactive material, resulting in enhanced antibody production and increasedimmune response. They seem to have the capability of concentrating theantigen and adjuvant on hydrophobic surfaces, where they are effectivelypresented to cells of the immune system.

Binding of antigen to a surface or presentation of a special type ofsurface for antigen adsorption, as in the case of emulsome-vaccines,appears to be critical for much of the biological activity of mostagents reported as adjuvants. Therefore the emulsome technology canserve as effective vehicles or delivery systems for human and veterinaryvaccines.

The use of emulsomes as a vaccine delivery system has other demonstrableadvantages. Emulsomes of this invention provide effective pharmaceuticaldelivery for a broad variety of both water-soluble and water-insolubleimmunogens with minimal local or systemic toxicity.

The hydrophobic core and surfactant provide a micro-environment whichaccommodates lipophilic immunogens such as lipid A orlipopolysaccharides as well as membrane-associated peptide antigendomains, while the aqueous continuous phase accommodates water-solublepeptide domains, or oligosaccharides.

The term "peptide" herein includes both oligopeptides and proteins. Tofacilitate intestinal uptake, the emulsions may be encapsulated ingelatin capsules or otherwise enterocoated to prevent their exposure togastric fluids when the oral route of administration is selected.

Additional advantages of the emulsome technology for delivery ofvaccines are: antigens and adjuvants can be incorporated simultaneouslyin the same formulation due to the hydrophilic-hydrophobic nature of theemulsome particles; high encapsulation efficiency of immunogens can beobtained; emulsomes can be formulated to act as depot for slow releaseof antigens avoiding the need for repeated vaccinations; themanufacturing technique of emulsomes is relatively simple and easy toscale-up.

The emulsome-vaccine formulations of this invention do not include anypolyoxypropylene-polyoxyethylene block polymer, trehalose dimycolate,cell wall skeleton, or any immunostimulatory mycobacteria or muramylpeptide-like additives to be effective.

Another aspect of this invention is to provide compositions and methodsfor the preparation of emulsomes containing antigens, incorporatedeither intrinsically (emulsified together with the phospholipids andsolid fat) or extrinsically (added externally to prepared emulsomes).

In some cases, the emulsomes of the present invention can beadministered in combination with other adjuvant systems, such asproteosomes, as indicated in the examples.

The size, concentration and specific composition of emulsome vaccinesmay be varied to suit the particular antigen used. Moreover, suchformulations may enhance both humoral and cell-mediated immunity (CMI)as do Freund's adjuvants. The emulsomes here described have beendeveloped for human use and since their particles are of submicron sizeand contain no added pyrogenic moieties such as mycobacteria ormuramyl-peptide derivatives they have, unlike Freund's adjuvants, greatsafety potential. They may be especially applicable to antigens that arevaccine candidates to protect against biologic toxins or infectiousagents which have natural hydrophobic moieties as a component includingtransmembrane viral, bacterial or parasite proteins, membrane proteinssuch as proteosomes, lipopolysaccharides, glycolipids such asgangliosides, or a variety of proteins or peptides to which hydrophobicanchors have been chemically or genetically added.

In addition to parenteral vaccination, another aspect of this inventionis to provide emulsome compositions and methods to achieve mucosalimmunity by using emulsome preparations comprising a plurality ofsubmicron particles, a mucoadhesive macromolecule, immunogenic peptideor antigen, and an aqueous continuous phase, which induces mucosalimmunity by achieving mucoadhesion of the emulsome particles to mucosalsurfaces. Mucous surfaces suitable for application of the emulsions ofthe present invention may include ocular (corneal, conjunctival), oral(buccal, sublingual), nasal, vaginal and rectal routes ofadministration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C are schematic illustrations of a liposome, anoil-in-water submicron emulsion droplet, and a proposed structure for anemulsome particle, respectively.

FIG. 2 is a graph showing the particle size distribution, asdifferential weight percent, of a group of emulsomes according to thepresent invention.

FIGS. 3A-C compares the ³¹ P-NMR spectra of submicron oil-in-wateremulsion, emulsomes, and liposomes, recorded in the absence ("A" series)and presence ("B" series) of PrCl₃ (30 mM).

FIG. 4 is a transmission electron micrograph of emulsome preparationnegatively stained with 1% phosphotungstic acid solution (Bar=100 nm).

FIG. 5 is a graph showing enhanced murine immunogenicity afterparenteral immunization of mice with formalinized SEB-Toxoid antigenformulated in intrinsic emulsome vaccine compared to free antigen oralum-adjuvanted vaccine.

FIG. 6 is a graph showing enhanced lapine immunogenicity of SEB-Toxoid Fantigen after parenteral immunization of rabbits with extrinsic emulsomevaccine compared to free antigen.

FIGS. 7A and 7B are a pair of graphs showing protection of miceimmunized with mucoadhesive emulsome vaccines containing SEB-Toxoid Fantigen or SEB-Tox F complexed to proteosomes against intranasalchallenge with SEB toxin in BALB/C mice D-galactosamine model.

FIG. 8 is a graph showing increased immune response in rhesus monkeysimmunized with anti-Leishmania intrinsic emulsome vaccine containingLC-467 lipopeptide antigen.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to pharmaceutical compositions for thedelivery of water-soluble and lipid-soluble vaccines, and to methods forpreparing and using such compositions.

As used herein, the term "lipid" refers to compounds which are solublein hydrocarbon solvents and are of a structural type which includesfatty acids and their esters, cholesterol and cholesteryl esters, andphospholipids.

The size range of emulsomes described in the present invention (10-250nm) makes them suitable for parenteral delivery. The 10-250 nm rangeincludes the mean size on a weight basis of preferred emulsomepreparations. In more preferred preparations, the 10-250 mn size rangeincludes at least 99% of the particles in the nanoemulsion, asdetermined on a weight basis. "Weight basis" determination as usedherein means that the weight percent rather than the number of the lipidparticles within the stated diameter range is used for the sizedetermination. In certain preparations, the mean particle size plus orminus the standard deviation falls within the range 20 to 180 nm, 40 to160 nm, or 50 to 150 nm. In other preparations, the mean and thestandard deviation falls within the range 10 to 120 nm. In still morepreferred preparations, 99% of the particles in the nanoemulsion fallwithin one of the above size ranges, as determined on a weight basis.All of the above emulsions can be sterilized by filtration.

Emulsomes can be administered parenterally, orally, topically, rectally,vaginally or intranasally.

Composition of the Lipid Core

An essential component of emulsomes is an internal hydrophobic or lipidcore comprising a lipid which exhibits solid or liquid crystal or mixedsolid and liquid crystal phases at room temperature (25° C.) whenmeasured in bulk. The lipid may be a single compound or a mixture. Theterm "lipid" as applied to the lipid core herein may refer either to asingle pure lipid compound or to a mixture of lipid compounds present inthe core.

Lipid compositions suitable for use as the core component of emulsomesmay be characterized as being in the solid or liquid crystalline phaseat at least about 25° C., when measured in bulk form withoutincorporation into emulsomes. Some lipid compounds present in a mixtureoptionally may be fluids at 25° C. when pure, provided that the lipidmixture as a whole is solid or liquid crystalline in bulk at 25° C. Inpreferred compositions, at least 90% of the individual lipid compoundspresent in the core are solids or liquid crystals at 25° C. whenmeasured in pure bulk form.

Phase determination preferably may be performed on the bulk lipid, i.e.,a macroscopic sample of the same composition, prior to its incorporationinto the emulsome core. The macroscopic phase determination on a bulksample may be made on a melting apparatus or by spectroscopic means,such as IR, NMR, or fluorescence intensity or anisotropy. Bulk phasedetermination of an existing emulsome preparation may be performed byfirst extracting the core lipids, then measuring.

Lipids which form the lipid core are composed almost exclusively ofnonpolar moieties which therefore do not exhibit a preference for thelipid-water interface. Triglycerides are the commonest type of fattyacid esters used in preparing the lipid core of nanoemulsions of thisinvention.

Triglycerides are a preferred material from which the lipid core may beprepared. The triglyceride core may be composed of a single puretriglyceride, usually available as a synthetic triglyceride, or may be amixture of several triglycerides. Fats isolated from natural sourcesusually are available only as mixtures of triglycerides. Such naturalmixtures are suitable for preparation of emulsomes, provided that themelting characteristics of the mixture are such that they exhibit asolid or liquid crystal phase at 25° C.

Detailed summaries of the phase behavior of various pure and mixedtriglycerides are available; see D. Small, "Glycerides," in: ThePhysical Chemistry of Lipids from Alkanes to Phospholipids, Chapter 10,Plenum Press, New York, 1985; and M. Kates, Techniques of Lipidology,Chapter 1, North Holland, Amsterdam/American Elsevier Publ. Co., Inc.,New York, 1972.

From the information available in these and other standard references,one skilled in the art may choose particular fats which have therequisite property of providing a solid or liquid crystal or mixed phaseat 25° C. when measured in bulk. The melting properties of particularmixtures of fats may be determined readily by simple experiments.

Many triglycerides which are solid at 25° C. have fully saturated fattyacid chains. Saturated fatty acids are advantageous because they areincapable of undergoing peroxidation reactions, which lessen theacceptable storage life of oil-in-water emulsions.

Examples of solid fats suitable for the preparation of emulsomes aretriglycerides composed of natural, even-numbered and unbranched fattyacids with chain lengths in the C10-C18 range, or microcrystallineglycerol triesters of saturated, even-numbered and unbranched fattyacids of natural origin such as tricaprin, trilaurin, trimyristin,tripalmitin, and tristearin. In general, any lipid component or mixtureof lipid components which provides a solid phase at room temperature(25° C.) when measured in bulk is suitable for the lipid core.

Other preferred lipid core components are esters of monounsaturatedfatty acids. Although monounsaturated fatty acids are capable ofundergoing peroxidation, they are less reactive than typicalpolyunsaturated fatty acids. Natural monounsaturated fatty acids havethe cis configuration. In general, these are lower melting thancompletely saturated fatty acid esters. Usually, therefore,monounsaturated fatty acid esters will be most useful in a mixture withhigher melting saturated fatty acid esters.

Other triglycerides which are solid at 25° C. include partiallyhydrogenated vegetable oils. Unlike naturally occurring unsaturatedfatty acids, hydrogenated oils contain unsaturated bonds in the transconfiguration, which is higher melting than the cis configuration.Partially hydrogenated vegetable oils yield solid vegetable shortening(e.g., CRISCO), which may be used to prepare emulsomes which are free ofcholesterol or cholesteryl esters.

Triglycerides containing polyunsaturated fatty acids may be present insmall amounts in the lipid core of emulsomes, provided that theresulting triglyceride mixture is in the solid or liquid crystal phaseat 25° C. when measured in bulk.

In some embodiments, the lipid of the hydrophobic core may have a solidto fluid phase transition (melting) temperature between 25° C. andphysiological temperature (37° C.) when measured in bulk. For example,tricaprin melts at 35°-37° C., and is wholly or predominantly in thefluid phase at physiological temperature.

Tricaprin may be used to form an excellent lipid core for nanoemulsions.The lipid core alternatively may be composed of lipids which are in thesolid phase at 37° C. when measured in bulk, such as higher saturatedtriglycerides, e.g., tripalmitin or tristearin.

Cores of mixed fluid and solid phases at 37° C. are also possible,particularly when the core contains mixtures of lipids.

The lipid or hydrophobic core of emulsomes also may be composed of orcontain monoesters of fatty acids, such as waxes. In general, waxes arelong chain fatty alcohol esters of fatty acids. Many waxes have suitablemelting characteristics for use in emulsomes, since they are solids at25° C. Examples include the esters from beeswax and spermaceti, such ascetyl palmitate.

Preferred waxes are made from saturated or monounsaturated fatty acidsand saturated or unsaturated fatty alcohols. An example of the latter isprovided by arachidyl oleate.

Other satisfactory monoesters include solid monoglycerides such asglyceryl monostearate, and fatty acid esters of short chain alcoholssuch as ethyl stearate.

Cholesterol and cholesteryl esters optionally may be incorporated intothe lipid core or the surrounding phospholipid envelope. Cholesterol andits esters change the packing structure of lipids, and in highconcentrations they induce the formation of a liquid crystal phase. Aliquid crystal phase may co-exist with a solid phase under someconditions.

Preferred cholesteryl esters are those of saturated or monounsaturatedlong chain fatty acids, such as palmitoyl or oleoyl, respectively.Cholesteryl esters may be present in levels up to 50 mol % relative tothe triglyceride or other solid lipid core component.

Since cholesterol has a polar alcohol group, it tends to incorporateinto the envelope monolayers or bilayers rather than into the lipid coreitself, and should be considered a component of the phospholipidenvelope rather than of the core.

The lipid cores of emulsome particles of this invention optionally maycontain one or more antioxidants. A preferred antioxidant isα-tocopherol or its derivatives, which are members of the Vitamin Efamily. Other antioxidants include butylated hydroxytoluene (BHT).

Antioxidants lessen the formation of oxidative degradation products ofunsaturated lipids, such as peroxides. The need for antioxidants may belessened by preparing the lipid core from saturated fatty acids.

Lipid particles of the invention preferably do not contain serumapolipoproteins such as apo B, apo AI, apo AII, or apo E. The apo Bprotein has the effect of targeting intravenously administered lipidparticles to certain cellular receptors, such as the LDL receptor onhepatocytes and certain other cells.

Lipid particles of the invention preferably also are substantially freeof intracellular marker proteins, such as those associated with theintracellular cytoskeleton (e.g., actin, myosin, troponin, tubulin,vimentin, spectrin).

Lipid particles which do not contain intracellular marker proteins orserum apolipoproteins are herein described as "noncellular" particles,since they lack characteristic indicia of lipid particles present in orderived from cellular sources.

In addition, preferred preparations of emulsomes are substantially freeof lipase and phospholipase enzymatic activity. As defined herein, anemulsion is "substantially free" of lipase or phospholipase activity ifthe emulsion lipids or phospholipids are enzymatically cleaved at a rateof less than 0.1% per day when stored at room temperature.

In addition to immunogens, other natural, synthetic, or recombinantproteins and peptides optionally may be present in emulsomes. An exampleof natural protein is collagen, which may be used to prepare emulsomeswith controlled or sustained release properties. This is described ingreater detail below.

Surface Active Molecules

In lipid particles of the invention, the lipid core is surrounded by atleast one envelope or layer containing phospholipid molecules. Thephospholipid envelope functions as a stabilizer or surface-active agentat the lipid-water interface, thereby lowering the surface tension.

In preferred embodiments, phospholipid molecules comprise at least 90%,more preferably 95%, even more preferably at least 99% of thesurface-active molecules covering the lipid core.

However, other surfactants may be used in small amounts, such as thenon-natural nonionic surfactant TWEEN. The lipid core of thenanoemulsion particles may be covered or surrounded by more than onelayer or envelope of surface-active molecules containing phospholipids.

In general, the surface-active phospholipid molecules are believed toform a monolayer around the lipid core of the particles, with the polarphospholipid head groups at the aqueous interface. However, particularlyat higher molar ratios of phospholipid to core lipid, excessphospholipid may be available to form one or more roughly concentricbilayers which encapsulate the lipid core with its associatedphospholipid monolayer. The number of bilayer envelopes is variable, andmay include one, two, or many bilayers. These bilayer envelopes entrapone or more aqueous compartments which may be made to contain awater-soluble antigen by creating the lipid particles in the presence ofan aqueous solution of that antigen.

Although the multiple concentric bilayer model of the structure ofemulsomes is proposed because it accounts for the observed ability ofthe particles to carry high loads of both lipid-soluble andwater-soluble antigens, the present invention does not depend upon andis not limited by the accuracy of the model.

Other geometric relationships between the lipid core and phospholipidmolecules are possible which might explain the antigen carrying capacityof emulsomes of the present invention.

The preferred phospholipids which constitute the surrounding envelopesof emulsomes are natural phospholipids such as soybean lecithin, egglecithin, phosphatidylglycerol, phosphatidylinositol,phosphatidyl-ethanolamine, phosphatidic acid, sphingomyelin,diphosphatidylglycerol, phosphatidylserine, phosphatidyl-choline,cardiolipin, etc.; synthetic phospholipids such asdimyristoylphosphatidylcholine, dimyristoyl-phosphatidylglycerol,distearoylphosphatidylglycerol, dipalmitoylphosphatidylcholine, etc.;and hydrogenated or partially hydrogenated lecithins and phospholipids.

In preferred embodiments, phospholipids which form "normal" phases(i.e., ionic "head" groups facing to the external aqueous phase andlipophilic "tails" facing internally) under physiological conditions ofpH and ionic strength comprise at least 50% of the total phospholipids,more preferably at least 75%, most preferably at least 90% on a molarbasis. Examples of normal phase forming phospholipids arephosphatidyl-choline (lecithin), phosphatidylglycerol, andphosphatidylinositol. By contrast, phosphatidlyl-ethanolamine has atendency to form reverse phases, with the polar head groups orientedinternally and the lipophilic tails oriented outwardly. Reverse phasesalso may be formed by cardiolipin or phosphatidic acid in the presenceof Ca⁺² ions; by phosphatidic acid at pH less than 3; and byphosphatidylserine at pH less than 4.

The phospholipid component may be either saturated or unsaturated, andmay have a gel to fluid phase transition temperature either above orbelow 25° C. Egg or soy phosphatidylcholines (egg or soy PC) areexamples of phospholipids with transition temperatures well below roomtemperature. Dimyristoyl phosphatidyl-choline (DMPC) has a transitiontemperature slightly below room temperature.

Dipalmitoyl and distearoyl phosphatidylcholines (DPPC and DSPC) areexamples of phospholipids with transition temperatures well above roomtemperature, and in fact even above physiological temperature (37° C.).Acceptable emulsomes may be made with these and many otherphospholipids.

In general, emulsomes prepared with phospholipids which are in the gelphase at 37° C. are expected to have more rigid bilayer envelopes andlonger circulation time in plasma.

Emulsomes may be prepared with molar ratios of phospholipid to totallipid in the range of 0.1 to 0.75 (10 to 75 mol %), more usually 0.1 to0.5 (10 to 50 mol %). The molar ratio of phospholipid to core lipidtypically may be in the range of 0.1:1 to 2:1, usually 0.1:1 to 1:1,often 0.2:1 to 0.9:1, frequently 0.2:1 to 0.8:1, and commonly 0.25:1 to0.6:1.

On a weight basis, the ratio of phospholipid to core lipid usually fallsin the range 0.5:1 to 1.5:1, and frequently 0.6:1 to 1.2:1.

Non-natural surfactants and detergents optionally may be incorporatedinto emulsomes in small amounts. As used herein, the terms "nonnaturalsurfactants" or "detergents" include a wide variety of manmade moleculeswhich form micelles in aqueous solution and contain both lipophilic andhydrophilic domains; however, phospholipids which belong to naturallyoccurring structural type are excluded from this definition, regardlessof whether a particular phospholipid is obtained by synthesis or byisolation from natural sources. Examples of non-natural surfactantsinclude the polysorbates ("TWEEN"), sodium dodecylsulfate (SDS),polyethoxylated castor oil ("CREMOPHOR"), NP-40, and numerous othersynthetic molecules. In preferred embodiments, nonnatural surfactantscomprise less than 10% (mol/mol) of the total surfactant, morepreferably less than 5%, still more preferably less than 1%, and mostpreferably less than 0.1%. A significant advantage of emulsomes is thatthey may be prepared as a stable nanoemulsion in the essential absenceof nonnatural surfactants. Even nonnatural surfactants which have beenapproved for parenteral administration are prone to cause toxic orundesirable side effects, whereas the phospholipid surfactants used inemulsomes are physiologically compatible.

In experiments to determine the effect of non-natural surfactants onemulsome structure, polysorbate (TWEEN-80) was added to an emulsomepreparation at final concentrations of 0.1, 0.5, and 1% (w/v). The meansize of the resulting emulsome particles decreased from 225 nm in theabsence of polysorbate to 120, 40, and 35 nm, respectively. Thusincreasing concentrations of synthetic surfactants progressivelydecrease the particle size, and higher concentrations than those usedare expected to result in formation of micelles (1-10 nm diameter).

Negatively charged lipid molecules such as oleic acid, or negativelycharged phospholipids such as phosphatidylglycerol, phosphatidic acid,phosphatidyl-inositol and phosphatidylserine, can be added to the lipidphase of emulsomes to increase the zeta potential of the composition,thus stabilizing the particles.

Additionally, the incorporation of these negatively charged lipidcompounds in emulsomes results in the formation of phospholipid bilayerswith opposing charges, thus increasing the loading of water-solublemolecules in the aqueous compartments formed by the phospholipidbilayers surrounding the lipid core. This effect results from the largeraqueous spaces between the bilayers caused by the electrostaticrepulsion between them. Another beneficial role of the inclusion ofnegatively charged lipid molecules in emulsomes is to reduce thelikelihood of particle aggregation, which minimizes destabilizingprocesses such as coalescence, flocculation, or fusion. Aggregation isprevented by the repulsive forces between the approaching particles.

Negatively charged phospholipids such as phosphatidylglycerol have beenincorporated into liposomal formulations used in human clinical studies;see, e.g., S. Amselem et al., J. Pharm. Sci. (1990) 79, 1045-1052; S.Amselem et al., J. Liposome Res. (1992) 2, 93-123. The significance ofzeta potential in analyzing and predicting the properties ofphospholipid bilayers is discussed in L. Sai-lung, Chapter 19, Vol. 1 in"Liposome Technology," 2nd ed., G. Gregoriadis, ed., CRC Press, BocaRaton, Fla. (1993), pp. 331-342. Both lipoidal particle size andparticle stability vary as a function of zeta potential. For liposomes,zeta potential and particle size increase in proportion to the contentof negatively charged phospholipid, up to 50 weight % of negativelycharged phospholipid.

The preferred range of negatively charged lipid in emulsome particles is0 to 30 mol % relative to total phospholipid and charged lipid, morepreferably 5 to 20 mol %, and still more preferably 7 to 15 mol %.

Incorporation of Immunogens

Emulsomes for use as vaccine vehicles contain an antigen of interest,usually an antigen bearing at least one epitope which is present on anorganism which is a pathogen in the animal species to be vaccinated. Inmost cases, the antigen is a peptide, a protein, or a glycoprotein.However, other antigenic structures may be employed, includingpolysaccharides, glycolipids, or a hapten conjugated to a carrier.

Since the emulsome particles provide a soluble or lipid-solubleimmunogens can be incorporated in emulsome vaccines of the presentinvention. Examples of peptide antigens are: hydrophilic natural orsynthetic peptides and proteins derived from bacteria, viruses andparasites, such as the recombinant gp160 envelope protein of the HIVvirus; natural or synthetic glycoproteins derived from parasites,bacteria or viruses such as the native surface glycoprotein ofLeishmania strain or subunit vaccines containing part of theglycopeptides alone or covalently conjugated to lipopeptides likelauryl-cystein hydrophobic foot; protein toxoids such as theStaphylococcus enterotoxin B toxoid, either chemically or physicallyinactivated; non-toxic bacterial surface structures (fimbrial adhesions)of Escherichia coli strains such as the Shiga-like Toxin B Subunit(SLT-B) and AF-R1, a pilus adhesion which is a virulence factor forRDEC-1 E. coli strain; outer membrane proteins of Neisseriameningitidis; Hepatitis B surface antigen; native or synthetic malariaantigens derived from different portions of Plasmodium falciparum, etc.Examples of lipophilic or hydrophobic immunogens are lipopolysaccharides(LPS), such as detoxified LPS obtained from E. coli (Sigma Chemical Co.,St. Louis, U.S.A.); Lipid A, the terminal portion of LPS, such as theone isolated from Salmonella minnesota R595 from List BiologicalLaboratories (California, U.S.A.).

In some embodiments, the emulsome particles will be free orsubstantially free of the above or other nonbioactive proteins, i.e.less than 5%, usually less than 1%, and frequently less than 0.1% (w/w)protein relative to other particle components.

In making a vaccine with emulsome vehicle, the antigen of interest maybe added to the organic solution of core lipid and phospholipid prior toforming a lipid film. The antigen is thereby integrally associated intothe structure of the lipid particles as they are formed. Alternatively,the antigen of interest may be present in the aqueous suspension oflipid particles prior to homogenization, or even added afterhomogenization. The latter method of preparation tends to produce moresuperficial binding of the antigen to the lipid particles.

Continuous Aqueous Phase

The aqueous component will be the continuous phase of the emulsomeformulation and may be water, saline or any other suitable aqueoussolution which can yield an isotonic and pH controlled preparation.

In addition, the compositions of the invention may also compriseconventional additives such as preservatives, osmotic agents or pressureregulators and antioxidants. Typical preservatives include Thimerosal,chlorbutanol, benzalkonium chloride, and methyl, ethyl, propyl or butylparabens. Typical osmotic pressure regulators include glycerol andmannitol, with glycerol being preferred. The preferred oil phaseantioxidant is tocopherol or tocopherol succinate. The aqueous phase mayalso include an antioxidant of a polyamine carboxylic acid such asethylene pharino tetra-acetic acid, or a pharmaceutically acceptablesalt thereof.

Mucoadhesive Emulsome Vaccines

Emulsome vaccine of the present invention optionally may contain abioadhesive macromolecule or polymer in an amount sufficient to conferbioadhesive properties. The bioadhesive macromolecule enhances thedelivery and attachment of antigens on or through the target mucoussurface conferring mucosal immunity. The bioadhesive macromolecule maybe selected from acidic non-naturally occurring polymers, preferablyhaving at least one acidic group per four repeating or monomeric subunitmoieties, such as polyacrylic acid and/or polymethacrylic acid (e.g.Carbopol, Carbomer), poly(methylvinyl ether/maleic anhydride) copolymer,and their mixtures and copolymers; acidic synthetically modified naturalpolymers, such as carboxymethylcellulose (CMC); neutral syntheticallymodified natural polymers, such as (hydroxypropyl) methylcellulose;basic amine-bearing polymers such as chitosan; acidic polymersobtainable from natural sources, such as alginic acid, hyaluronic acid,pectin, gum tragacanth, and karaya gum; and neutral non-naturallyoccurring polymers, such as polyvinylalcohol; or their mixtures.

The ionizable polymers may be present as free acids, bases, or salts,usually in a final concentration of 0.01-0.5% (w/v).

As used herein, a polymer of an indicated monomeric subunit contains atleast 75%, preferably at least 90%, and up to 100% of the indicated typeof monomer subunit; a copolymer of an indicated type of monomericsubunit contains at least 10%, preferably at least 25% of that monomericsubunit.

A preferred bioadhesive macromolecule is the family of acrylic acidpolymers and copolymers (e.g. CARBOPOL™). These contain the generalstructure:

    -- --CH.sub.2 --CH(COOH)--!-n

One preferred group of polymers of acrylic acid is commerciallyavailable under the tradename Carbopol. Carbopol 934 is available in apharmaceutical grade.

Preferred bioadhesive or mucoadhesive macromolecules have a molecularweight of at least 50 kDa, preferably at least 300 kDA, and mostpreferably at least 1,000 kDa. Favored polymeric ionizable mucoadhesivemacromolecules have not less than 2 mole percent acidic groups (e.g.COOH, SO₃ H) or basic groups (NH₂, NRH, NR₂), relative to the number ofmonomeric units. More preferably, the acidic or basic groups constituteat least 5 mole percent, more preferably 25 or even 50, up to 100 mole %relative to the number of monomeric units of the macromolecule.

Preferred macromolecules also are soluble in water throughout theirrelevant concentration range (0.01-0.5% w/v).

Polymeric Coating of Emulsomes

Biodegradable polymers may be incorporated to surround or form part ofthe hydrophobic core of emulsomes. Polymeric emulsomes may containbiodegradable nonnatural polymers such as polyesters of lactic andglycolic acids, polyanhydrides, polycaprolactones, polyphosphazenes andpolyorthoesters, or natural polymers such as gelatin, albumin, andcollagen. The advantage of polymeric emulsomes is to provide controlledrelease for the parenteral delivery of drugs and biological compounds ina sustained dosage form.

The structure, selection, and use of degradable polymers in drugdelivery vehicles have been reviewed in a recent publication (A. Domb,S. Amselem, J. Shah and M. Maniar, Polymers for Advanced Technologies(1992) 3, 279-292). Further guidance to selection of polymers isavailable in any standard text on that topic.

In general, the ratio of polymer to lipid core (e.g., triglyceride) maybe up to 50% (w/w). For the natural protein polymers such as gelatin,which swell extensively in aqueous solution, useful levels ofencapsulation may be achieved with much lower amounts of polymer, suchas 1% to 10% (w/w).

For most non-natural polymers, which are soluble in organic solvents,the polymer may be codissolved with the triglyceride and phospholipidprior to the evaporation step. For natural polymers which are soluble inaqueous solution, the polymer may be dissolved in the solution used.

Dehydrated Emulsomes

A further aspect of the invention provides dehydrated emulsomes, made bydehydrating the compositions of the types described herein. Dehydrationmay be performed by standard methods, such as drying under reducedpressure; when the emulsion is frozen prior to dehydration, this lowpressure evaporation is known as lyophilization. Freezing may beperformed conveniently in a dry ice-acetone or ethyl alcohol bath. Thepressure reduction may be achieved conveniently with a mechanical vacuumpump, usually fitted with a liquid nitrogen cold trap to protect thepump from contamination. Pressures in the low millitorr range, e.g.10-50 millitorr, are routinely achievable but higher or lower pressuresare sufficient.

Emulsomes can be lyophilized by adding cryoprotectants such as sugars oramino acids, and stored as freeze-dried solid material that can bereconstituted with the aqueous medium before us, thus conferring furtherstability of the incorporated antigens.

Preferred cryoprotectants include sugars such as glucose, sucrose,lactose, maltose, and trehalose; polysaccharides such as dextrose,dextrins, and cyclodextrins; nonnatural polymers such aspolyvinylpyrrolidone (PVP); and amino acids. The preferred range ofcryoprotectant to emulsome phospholipid is 0.1% up to 10% (w/w).

Dehydration of an emulsome nanoemulsion yields a solid residue which maybe stored for prolonged periods, and may be rehydrated to yield anemulsome nanoemulsion having an average particle size similar to that ofthe original nanoemulsion. The dehydrated emulsomes also retainsubstantial amounts of the originally incorporated antigen.

The amount of water remaining in the dehydrated emulsome preparation mayvary widely depending upon the type and extent of dehydration procedureemployed. In general, the dehydrated emulsomes contain less than 1%water by weight, especially when dehydrated by lyophilization. However,stable dehydrated preparations may contain up to 5% water.

Dry compositions for preparing submicron emulsions are disclosed indetail in PCT International Application No. PCT/US93/01415, which wasfiled on 17 Feb. 1993, and published as WO 93/15736 on 19 Aug. 1993. Thedisclosure of said document is expressly incorporated herein in itsentirety by this reference thereto.

Distinctive Features of Emulsomes

Emulsomes of this invention are distinct from standard oil-in-wateremulsions. Due to the high phospholipid content of the currentinvention, a monolayer of phospholipid surrounds the lipid core at theaqueous interface thereby stabilizing the emulsion. In addition, one ormore bilayers or envelopes of phospholipid molecules are believed toform around the particles in many embodiments. Another major differenceis that while standard oil-in-water emulsions are dispersions of oneliquid into another, emulsomes are dispersions of a solid in a liquid.

The main differences between oil-in-water emulsions and emulsomes aresummarized in Table 1.

One major drawback of standard oil-in-water emulsions is limited drugloading. When drug encapsulation above 1% is required, a correspondinglylarger oil phase (10-20%) is required to dissolve the drug. However, thehigh oil content reduces the stability of the emulsion, and the additionof a surfactant or cosurfactants, is necessary. Due to the detergentproperties of most surfactant compounds, their use for parenteraladministration is very limited. Many toxic reactions have been reportedeven with the surfactants already approved for parenteral formulations,such as sodium deoxycholate, poloxamer-188 (Pluronic F-68), polysorbate80 (TWEEN 80), and Emulphor EL-620 or polyethoxylated castor oil.

The pharmaceutically stable emulsomes described herein have majoradvantages over standard emulsions in that water-soluble and waterinsoluble antigens can be encapsulated either separately orsimultaneously at high loadings in the absence of any nonnatural ionicor non-ionic surfactant.

                  TABLE 1                                                         ______________________________________                                                               Emulsome                                                                      Dispersion                                                                              Liposome                                                 SME        of a solid                                                                              Dispersion                                               Dispersion fat or    of phospho-                                              of an oil  lipid in  lipids in                                    Definition  in water   water     water                                        ______________________________________                                        Internal core                                                                             oil        solid or  water                                                               liquid                                                                        crystalline                                                                   lipid                                                  Phospholipid                                                                              0.5-2%     5-10%     0.1-5%                                       content (w/v)                                                                 Non-natural present    usually   usually                                      surfactant             absent    absent                                       Co-surfactant                                                                             present    usually   usually                                                             absent    absent                                       Lipophilic  up to      up to     up to                                        loading     10 mg/ml   100 mg/ml 20 mg/ml                                     PC/total lipid                                                                            0.01-0.1   0.1-0.5   0.6-1.0                                      (mol/mol)                                                                     ______________________________________                                    

Emulsomes of this invention differ from the microdroplets of U.S. Pat.Nos. 4,725,442 and 4,622,219. Microdroplets, originally called monolayervesicles, consist of spheres of organic liquid covered by one monolayerof phospholipid, while the internal core of emulsomes consists of asolid lipid or fat. The phospholipid content of microdroplets is low(about 1.2%) forming only one monolayer, while in emulsomes thephospholipid content is high (5-10%) and in certain embodiments isbelieved to form several bilayers surrounding the fat core. Anothermajor difference between microdroplets and emulsomes is thatmicrodroplets are useful only for water-insoluble compounds, while inemulsomes, due to the high lecithin content, water-soluble as well aswater-insoluble compounds can be incorporated.

Methods of Preparation of Emulsomes

A further embodiment of the invention relates to methods for preparationof emulsome vaccines intrinsically and extrinsically as extensivelydetailed in the examples. In general, emulsome intrinsic formulationsare prepared by emulsifying the antigen together with the emulsomecomponents, while emulsome extrinsic formulations are prepared by addingexternally the antigen to previously prepared plain emulsomes.

Emulsomes may be prepared by mixing phospholipids and triglycerides in aweight ratio range of 0.5:1 wherein the triglyceride has a solid toliquid phase transition temperature of greater than 25° C.; suspendingthe mixture in an aqueous solution at a temperature below the solid toliquid transition temperature of the triglyceride; and homogenizing orotherwise reducing the suspension to yield the emulsomes. Theseemulsomes comprise a nanoemulsion of lipid particles having a meanparticle diameter of between about 10 nm and 250 nm, usually within therange 20 to 180 nm, and frequently within the range 50 to 150 nm.

These size ranges preferably are determined on a weight percent basis,rather than a particle number basis. The cited ranges include the meanparticle size. In certain embodiments, the cited ranges include the meanplus or minus the standard error, and in other embodiments the citedranges include at least 99% of the particles as determined on a weightbasis.

Conveniently, the lipid components may be dissolved in a volatile andchemically unreactive organic solvent such as dichloromethane or diethylether. The organic solvent is removed, typically under reduced pressurein a rotary evaporator or under a stream of inert gas. The resultinglipid film is hydrated and dispersed by covering and shaking with anaqueous solution. The immunogen to be incorporated according to itschemical properties and hydrophilic-lipophilic nature can be included inthe lipid phase or may be added to the aqueous hydration solution.

Water-soluble antigens are encapsulated or entrapped in emulsomes bydissolving them in the aqueous medium, hydrating the dryfat-phospholipid mixture with the aqueous phase containing the antigenutilizing mechanical shaking, and sizing the resultant dispersion byhigh shear-homogenization to the desired final size range.

Lipid-soluble antigens may be incorporated into solid lipidnanoemulsions by dissolving them in a suitable organic solvent togetherwith the lipid ingredients of the composition, e.g., phospholipids andsolid fatty acid esters, evaporating the solvent to complete dryness,hydrating the antigen-lipid mixture with the aqueous phase utilizingmechanical shaking, and homogenizing the resultant dispersion withhigh-shear homogenizers to final sizes in the range of 10 to 250 nm.

The lipid suspension or dispersion is then sized, typically by highshear homogenization at pressures up to 800 bar in a Gaulin-typehomogenizer (AVP Gaulin International, Holland) or EmulsiFlex™homogenizer (Avestin Inc., Canada). High pressure Gaulin homogenizationis described in detail in Brandl et al., in Liposome Technology, 2nded., G. Gregoriadis, ed., Vol. 1, Ch. 3, CRC Press, Boca Raton, Fla.,(1993), pp. 49-65.

Emulsomes also may be prepared by high pressure extrusion throughpolycarbonate membranes. In this procedure, the sizing step on the lipiddispersion is performed using a pressure extruder, such as the stainlesssteel GH76-400 Extruder or Pressure Cell (Nucleopore, U.S.A.), ratherthan a high-shear homogenizer. The pressure extruder and the extrusiontechnique for liposome preparation are described in detail in S. Amselemet al., in Liposome Technology, 2nd ed., G. Gregoriadis, ed., Vol. 1,Ch. 28, CRC Press, Boca Raton, Fla., (1993), pp 501-525.

Due to the small size of emulsomes they can be sterilized by finalsterile filtration through 0.2 μm filter membranes.

EXAMPLES

This invention is illustrated by the following non-limiting examples:

Example 1 PREPARATION OF EMULSOMES USING A HIGH SHEAR MICROLAB 70 GAULINHOMOGENIZER

To a 0.5 liter round-bottomed flask, 2.5 g of egg-lecithin, 2.5 g oftricaprin, 0.1 g of cholesterol, 0.1 g of oleic acid and 0.01 g oftocopherol succinate were added. The lipid mixture was dissolved in 50ml dichloromethane. The organic solvent was evaporated until completedryness under reduced pressure using a rotary evaporator (Heidolph,Germany). To the dry lipid film 50 ml of saline were added and themixture was then hydrated by shaking until all the lipids werehomogeneously dispersed in the aqueous phase. The dispersion washomogenized for five minutes at 15,000 rpm using a Polytron PT 3000(Kinematica, AG). The preparation was then submitted to 10-15 cycles ofhigh shear homogenization at 800 bar using a Microlab 70 GaulinHomogenizer (AVP Gaulin International, Holland). The particle sizedistribution of the formulation was determined using a N4MD CoulterParticle Size Analyzer (Coulter Electronics, England). The differentialweight % mode of the instrument indicated the existence of a singlehomogeneous population of emulsomes with a mean particle diameter of84±32 nm. The formulation was shown to be stable at room temperature forseveral months without changes in the mean size of the particles.

Example 2 PREPARATION OF EMULSOMES USING A EMULSIFLEX™ HOMOGENIZER

To a 0.5 liter round-bottomed flask, 3.5 g of egg-lecithin, 3.5 g oftricaprin, 0.2 g of cholesterol, 0.2 g of oleic acid, and 0.05 g oftocopherol succinate were added. The lipid mixture was dissolved in 50ml dichloromethane. The organic solvent was evaporated until completedryness under reduced pressure using a rotary evaporator (Heidolph,Germany). To the dry lipid film 70 ml of saline were added and themixture was then hydrated by shaking until all the lipids werehomogeneously dispersed in the aqueous phase (estimated hydration time,1 hr). The dispersion was homogenized by submitting the preparation to5-7 cycles of high shear homogenization at 12,500 psi working pressureusing a EmulsiFlex™ C30 Homogenizer (Avestin Inc., Canada). The particlesize distribution of the resultant emulsomes was determined using a N4MDCoulter Particle Size Analyzer (Coulter Electronics, England). Thedifferential weight % mode of the instrument indicated the existence ofa single homogeneous population of emulsomes with a mean particlediameter of 109±30 nm. FIG. 2 shows the particle size distribution ofemulsomes prepared by EmulsiFlex™ instrument.

Example 3 CHARACTERIZATION OF EMULSOME PARTICLES

The parameters most frequently used to characterize lipid particles areparticle size and size distribution, morphology, and lamellarity.Emulsome structure was demonstrated and characterized by severaltechniques including electron microscopy, photon correlationspectroscopy, and NMR.

Particle Size Distribution:

The particle size distribution of the emulsome formulations weredetermined by photon correlation spectroscopy (PCS), based on measuringlaser scattered-light fluctuations, using a N4MD Coulter SubmicronParticle Size Analyzer (Coulter Electronics, England) working at thedifferential weight % operation mode of the instrument (Barenholz, Y.,and Amselem, S. In "Liposome Technology", Gregoriadis G., ed., 2ndedition, Vol. 1, pp 527-616, CRC press, Florida, 1993).

The particle size distribution of all emulsome formulations described inthe Examples was determined by photon correlation spectroscopy using theN4MD Coulter. The N4MD Coulter submicron particle analyzer, indicatedthe existence of a single homogeneous population of Emulsomes with amean particle diameter in the range of 50-200 nm (FIG. 2).

³¹ P-NMR measurements:

One of the most accurate and straightforward procedures forquantitatively determining the lamellarity of phospholipid dispersionsis to use NMR spectroscopy and especially ³¹ P-NMR signal to monitor thephospholipid phosphorous signal intensity (Lichtenberg, D., Amselem, S.,and Tamir, I. Biochemistry, 18, 4169-4172, 1979). In particular, addingan impermeable paramagnetic shift reagent to the external medium willdecrease the intensity of the initial ³¹ P-NMR signal by an amountproportional to the fraction of lipid exposed to the external medium.FIG. 3 shows phosphorous nuclear magnetic resonance (³¹ P-NMR) spectraof liposomes, emulsomes, and submicron oil-in-water emulsion recordedbefore and after the addition of the lanthanide ion Pr⁺³. The ³¹ P-NMRspectra were recorded on a Brucker AM400 instrument employing a 50 KHzsweep width, 3 sec interpulse delay and broadening proton decoupling.Phosphoric acid in D₂ O was used as standard for 0 ppm chemical shift.

For the submicron oil-in-water emulsion (SME) after the addition of theparamagnetic ion the whole ³¹ P-NMR signal was shifted downfield (FIG.3B1). This result is expected since in the SME all the phospholipidmolecules are located in the surface of the emulsion oil droplets as amonolayer, thus the Pr⁺³ ions interact with all the exposed phospholipidmolecules. On the other hand for emulsomes, the ³¹ P-NMR signal wassplit into two peaks after Pr+3 addition (FIG. 3B2), indicating thatonly a portion of the phospholipid molecules (those in the outermonolayer exposed to the aqueous medium) interacted with the Pr⁺³ ionsdemonstrating the existence of bilayer structures. The same picture wasobtained for small unilamellar liposomes (FIG. 3B3) used as control,where the existence of phospholipid bilayer domains have been welldocumented (Barenholz, Y., and Amselem, S. In "Liposome Technology",Gregoriadis G., ed., 2nd edition, Vol. 1, pp. 527-616, CRC press,Florida, 1993).

Electron microscopy:

Negative stain electron microscopy (EM) has been used to characterizethe size and shape of the lipid particles present in phospholipiddispersions. EM gives also a gross estimate of lamellarity since stainpenetrates interbilayer spaces and allows lamellae to be resolved. FIG.4 is an transmission electron micrograph of emulsome preparation showingspheric particles having a diameter in the range of 80-130 nm surroundedby well-defined phospholipid bilayers enveloping the internalhydrophobic core made of triglycerides.

Example 4 PREPARATION OF EXTRINSIC MUCOADHESIVE EMULSOME VACCINE

To a round 0.5 liter round-bottomed flask, 1.75 gr of egg-lecithin, 1.75gr of tricaprin, 70 mg of cholesterol, 70 mg of oleic acid, and 7 mg oftocopherol succinate were added. The lipid mixture was dissolved in 50ml of chloroform. The organic solvent was evaporated until completedryness under reduced pressure using a rotary evaporator (Heidolph,Germany). To the dry lipid film 60 ml of aqueous solution containing0.1% EDTA were added and the mixture was then hydrated by shaking or 30min. using a multiwrist shaker (Labline, U.S.A.) until all the lipidswere homogeneously dispersed in the aqueous phase. The dispersion washomogenized using a Microlab 70 Gaulin Homogenizer (5 cycles at 800bar). The particle size distribution of the resultant emulsomes wasdetermined using a N4MD Coulter Particle Size Analyzer (CoulterElectronics, England). The indicated the existence of a singlehomogeneous population of emulsomes with a mean particle diameter of140±50 nm. Then 6.72 gr of a 1% Carbopol solution was added and stirredfor 20 min to confer mucoadhesive properties to the emulsomepreparation. Glycerol (1.44 gr) were added thereafter to reach aphysiological osmolarity (269 mOsm). The pH was adjusted to 6.0 using a1M NaOH solution. To this plain mucoadhesive emulsome preparation,antigens can be added extrinsically and mixed with the emulsome carrierparticles by gentle shaking in order to obtain the proper emulsomevaccine.

Example 5 PREPARATION OF INTRINSIC HEPATITIS B EMULSOME VACCINE

To a round 0.25 liter round-bottomed flask, 2.5 gr of egg-lecithin, 2.5gr of tricaprin, 100 mg of cholesterol, 100 mg of oleic acid, and 10 mgof tocopherol succinate were added. The lipid mixture was dissolved in25 ml of chloroform. The organic solvent was evaporated until completedryness under reduced pressure using a rotary evaporator (Heidolph,Germany). To the dry lipid film 60 ml of phosphate buffered salinecontaining 0.5 mg of Hepatitis B antigen were added and the mixture wasthen hydrated by shaking for 30 min using a Multiwrist shaker (Labline,U.S.A.) until all the lipids were homogeneously dispersed in the aqueousphase. The dispersion was homogenized using a Microlab 70 GaulinHomogenizer (5 cycles at 800 bar). The particle size distribution of theresultant emulsomes was determined using a N4MD Coulter Particle SizeAnalyzer (Coulter Electronics, England). The differential weight % modeof the instrument indicated the existence of a single homogeneouspopulation of emulsomes with a mean particle diameter of 105+24 nm. Theemulsome vaccine formulation was then 2-fold concentrated using aFiltron ultrafiltration stirred cell (Omega Series membrane with 10,000molecular weight cutoff, Filtron Technology Corp., Massachusettes).

Example 6 PREPARATION OF MUCOADHESIVE INTRINSIC ANTI-HIV ENVELOPEPROTEIN (qp160) EMULSOME VACCINE

Antigen description and background: The urgency and high priority fordeveloping an effective vaccine against the human immunodeficiency virus(HIV) are fully recognized. The reasons for using subunits of the virusas the basis of an HIV vaccine are the perceived overwhelmingrequirements for safety. Despite the high efficacy of many liveattenuated viral vaccines, the requirement for product safety,especially in the case of retroviruses, has favored the subunit approachto the extent that all of the current candidate preparations in clinicalprophylactic trials are of this type, being mainly gp160, the envelopeprotein of HIV, or part thereof. Studies have shown that gp160 attachesthe virus to the cell and also facilitates the fusion of the cell andvirus during the early stages of infection.

Emulsome preparation:

To a round 0.25 liter round-bottomed flask, 0.24 gr of egg-lecithin,0.24 gr of tricaprin, 20 mg of cholesterol, and 20 mg of oleic acid, and2 mg of tocopherol succinate were added. The lipid mixture was dissolvedin 50 ml of chloroform. The organic solvent was evaporated untilcomplete dryness under reduced pressure using a rotary evaporator(Heidolph, Germany). To the dry lipid film 60 ml of aqueous solutioncontaining 0.18 mg of gp160 antigen and 0.1% EDTA were added and themixture was then hydrated by shaking for 30 min. using a multiwristshaker (Labline, U.S.A.) until all the lipids were homogeneouslydispersed in the aqueous phase. The dispersion was homogenized using aMicrolab 70 Gaulin Homogenizer (6 cycles at 800 bar). The particle sizedistribution of the resultant emulsomes was determined using a N4MDCoulter Particle Size Analyzer (Coulter Electronics, England). Thedifferential weight % mode of the instrument indicated the existence ofa single homogeneous population of emulsomes with a mean particlediameter of 158±57 nm. The emulsome vaccine formulation was then 5-foldconcentrated using a Filtron ultrafiltration stirred cell (Omega Seriesmembrane with 10,000 molecular weight cutoff, Filtron Technology Corp.,Massachusetts). Then 1.3 gr of a 1% Carbopol solution was added andstirred for 15 min to confer mucoadhesive properties to the emulsomevaccine preparation. Glycerol (0.285 gr) were added thereafter to reacha physiological osmolarity. The pH was adjusted to 6.0 using a 0.5M NaOHsolution. The estimated final gp160 concentration in the formulation was15 μg/ml.

Example 7 PREPARATION OF MUCOADHESIVE INTRINSIC EMULSOME VACCINECONTAINING ANTI-HIV ENVELOPE PROTEIN (gp160) COMPLEXED TO PROTEOSOMES

Proteosomes are meningococcal outer membrane protein preparationspurified from Neisseria meningitidis by detergent extraction andammonium sulphate precipitation. They naturally form 20-100 nm diameterhydrophobic membranous vesicles. Antigens are non-covalently complexedto proteosomes via hydrophobic interactions by mixing the antigen andproteosomes in the presence of detergent and then removing the detergentover a prescribed period of time, permitting hydrophobic interactions tooccur in the system.

Proteosomes have previously been shown to enhance the parenteralimmunogenicity of peptides, gangliosides, lipopolysaccharides andproteins hydrophobically complexed to them (Lowell, G. H., L. F. Smith,R. C. Seid and W. D. Zollinger, J. Exp. Med. 167, 658-663, 1988).(Lowell, G. H., W. R. Ballou, L. F. Smith, R. A. Wirtz, W. D. Zollingerand W. T. Hockmeyer. Science 240, 800-802, 1988; Lowell, G. H. 1990. In:"New Generation Vaccines". G. C. Woodrow and M. M. Levine (eds.), MarcelDekker, Inc., New York, p. 141-160) and have been shown to be safe forhuman use in vaccine trials involving tens of thousands of humans in thedevelopment of anti-meningococcal vaccines (Zollinger, W. D. New andImproved Vaccines Against Meningococcal Disease. In: "New GenerationVaccines", G. C. Woodrow and M. M. Levine (eds.), Marcel Dekker, Inc.,New York, p. 325-348). Furthermore, proteosomes confer mucosalimmunogenicity upon non-immunogenic antigens when administered orally orintranasally. Such intranasal or oral proteosome vaccines induce up to100% protection against lethal pneumonia or keratoconjunctivitis inexperimental murine models of shigellosis (Orr, N., G. Robin, D. Cohen,R. Arnon and G. Lowell. 1993. Immunogenicity and efficacy of oral orintranasal Shigella flexneri 2a and Shigella sonneiproteosome-lipopolysaccharide vaccines in animal models. Infect. Immun.61, 2390-2395).

Lipid and aqueous phases were prepared as described in Example 6. A vialcontaining 0.18 mg of gp160 non-covalently complexed to proteosomes andsuspended in saline was added to the water phase (60 ml total volume)and the mixture was gently shaken for 5 min. The subsequent stepsinvolved in the preparation of the mucoadhesive emulsome vaccine werecarried out as described in Example 6. The particle size volume %distribution of the resultant emulsome formulation showed a mean dropletsize of 112 nm. The estimated final gp160 concentration in theformulation was 15 μg/ml.

Example 8 PREPARATION OF MUCOADHESIVE EXTRINSIC EMULSOME VACCINECONTAINING gp160 ALONE OR COMPLEXED TO PROTEOSOMES

Mucoadhesive extrinsic emulsome formulation containing gp160 alone orgp160 complexed non-covalently to proteosomes was performed by preparingplain emulsomes as described in Example 7, but in the absence of theantigen and adding externally an aqueous dispersion of the gp160 orgp160-conjugated to proteosomes to the plain emulsomes by gently shakingfor 15 min at room temperature.

Example 9 PREPARATION OF INTRINSIC EMULSOME VACCINE CONTAININGSTAPHYLOCOCCUS ENTEROTOXIN B TOXOID-F ANTIGEN

Antigen description and background: Staphylococcal enterotoxin B (SEB)is a potent toxin that causes food borne illness among civilians andmilitary personnel stationed around the world and is identified as alethal offensive military threat that endangers both military andcivilian populations through aerosolization.

SEB infection in civilian populations is related to staphylococcal foodpoisoning by SEB and related toxins: also contributes to death fromstaphylococcal sepsis following overwhelming staph infection. It alsocauses staph scalded skin syndrome in kids--i.e. morbidity and mortalityfrom staphylococcal infections (P. Marrack and J. Kappler, Science,248,705-711, 1990).

Due to the similarity to the human response both in sensitivity andclinical signs and the lack of an established model for lethality to SEBdelivered via the respiratory route in lower animal species, non-humanprimates have been the primary animal model for development of vaccinesto protect against respiratory challenge with SEB. Early work indicatedthat monkeys develop decreased sensitivity to repeated mucosaladministration of the toxin. This suggested that protection to SEBexposure might be provided by toxoid immunization. Studies in rhesusmonkeys and other animals indicated that oral immunization withformalinized toxoid was ineffective against parenteral challenge whereasparenteral immunization with formalinized SEB toxid induced serumantibodies that recognized native SEB (Bergdoll, M. S. Enterotoxins. pp.559-598 In: Staphylococci and Stap hylococcal Infections, eds. C. S. F.Easmon and C. Adlam, Academic Press, London, 1983). In the latterstudies, however, several parenterally immunized monkeys that acquiredanti-SEB antibodies had severe immediate-type hypersensitivity reactionswhen challenged with SEB toxin. These adverse reactions suggested thatthe formalinized SEB toxoid alone was not a candidate for parenteralvaccine development. Additionally, as the military threat would be byaerosolization, it was determined that studies on protection provided byserum IgG to respiratory challenge as well as protective effectsprovided by anti-SEB secretory IgA in the respiratory tract wererequired.

Recently, two identical lots of formalinized SEB toxoid were made atWalter Reed Army Institute of Research, Washington DC (WRAIR) followingpreviously described specifications (Kaminski, R., S. Grate, E.Aboud-Pirak, C. Hooper, T. Levin, I. Weiss, S. Amselem, R. Arnon and G.Lowell, 1993. In: Proceedings of 1993 Medical Defense Bioscience Review,Baltimore, Md.). This WRAIR formalinized toxoid preparation designatedTox-F was non-toxic in rabbits at 0.5 mg/kg, the dose at which SEB toxinis invariably lethal. Furthermore, it was non-toxic in the murineD-galactosamine model of SEB toxicity even at 500 μg per BALB/c mouse;50 μg of SEB is 100% lethal in such mice. The physical characteristicsof Tox-F were similar to that described by Eldridge (Eldridge, J. H.,Staas, J. K., Meulbroek, J. A., Tice, T. T. and Gilley, R. M.Biodegradable and biocompatible poly(DL-lactide-co-glycolide)microspheres as an adjuvant for staphylococcal enterotoxin B toxoidwhich enhances the level of toxin-neutralizing antibodies. (Infect.Immun. 59, 2978-2986, 1991) in that SDS-PAGE gel of Tox-F showed twodistinct bands with estimated MW of 23,000 and 46,000. Biologically,Tox-F also had the characteristics previously reported by Eldridge etal., namely in a Mouse Spleen Lymphocyte Proliferative Assay in whichconcentrations of SEB toxin of 0.37-10.0 μg/ml were mitogenic, Tox-F wasentirely non-mitogenic at all concentrations tested (0.04-100.0 μg/ml).

Preparation of SEB-Toxoid F:

Formalinized SEB-Toxoid (Tox-F) was prepared according to the method ofWarren, J. R., Spero, L. and Metzger, J. F. 1983. J. Immunol. 111,885-892 and as per Eldridge, J. H. et al. 1991, Infect. Immun. 59,2978-2986 by formalin treatment for 30 days at 37 C., pH 7.5.

Preparation of Emulsomes:

To a round 0.5 liter round-bottomed flask, 2.25 gr of egg-lecithin, 2.25gr of tricaprin, 90 mg of cholesterol, 90 mg of oleic acid, and 9 mg oftocopherol succinate were added. The lipid mixture was dissolved in 50ml of chloroform. The organic solvent was evaporated until completedryness under reduced pressure using a rotary evaporator (Heidolph,Germany). To the dry lipid film 50 ml of aqueous solution containing 10mg of Staphylococcus Enterotoxin B toxoid, glycerol 2.25% and 0.1% EDTAwere added and the mixture was then hydrated by shaking until all thelipids were homogeneously dispersed in the aqueous phase. The dispersionwas homogenized using a Microlab 70 Gaulin Homogenizer (6 cycles at 800bar). The particle size distribution of the resultant formulation wasdetermined using an N4MD Coulter Particle Size Analyzer (CoulterElectronics, England). The differential weight % mode of the instrumentindicated the existence of a single and homogenous population ofemulsome particles with a mean particle size distribution of 115±80 nm.The estimated final antigen concentration in the formulation was 220μg/ml.

Example 10 PREPARATION OF EXTRINSIC EMULSOME VACCINE CONTAININGSTAPHYLOCOCCUS ENTEROTOXIN B-TOXOID-F

Extrinsic formulation of SEB-Toxoid-F in emulsomes was performed bypreparing plain emulsomes as described in Example 9, but in the absenceof the antigen and adding externally the aqueous solution containing theSEB-Toxoid-F to the plain emulsomes by gently shaking for 15 min at roomtemperature. A total volume of 0.78 ml of stock plain emulsomes weremixed with 0.78 ml solution of SEB-Toxoid-F (1 mg/ml protein) in 0.15MNaCl and 0.01M Tris buffer to give a final SEB-Toxoid-F concentration of0.5 mg/ml.

Example 11 PREPARATION OF INTRINSIC EMULSOME VACCINE CONTAININGSTAPHYLOCOCCUS ENTEROTOXIN B-TOXOID-F COMPLEXED TO PROTEOSOMES

To a round 0.5 liter round-bottomed flask, 2.5 gr of egg-lecithin, 2.5gr of tricaprin, 100 mg of cholesterol, and 100 mg of oleic acid, and 10mg of tocopherol succinate were added. The lipid mixture was dissolvedin 50 ml of chloroform. The organic solvent was evaporated untilcomplete dryness under reduced pressure using a rotary evaporator(Heidolph, Germany). To the dry lipid film 50 ml of aqueous solutioncontaining 5 mg of Staphylococcus Enterotoxin B toxoid-F complexed toproteosomes, and glycerol (2.25% w/v) were added and the mixture wasthen hydrated by shaking until all the lipids were homogeneouslydispersed in the aqueous phase. The dispersion was homogenized using aMicrolab 70 Gaulin Homogenizer (5 cycles at 800 bar). The particle sizedistribution of the resultant formulation was determined using an N4MDCoulter Particle Size Analyzer (Coulter Electronics, England). Theunimodal mode of the instrument indicated the existence of a single andhomogenous population of emulsome particles with a mean particle sizedistribution of 152±53 nm. The estimated final antigen concentration inthe formulation was 100 μg/ml.

Example 12 PREPARATION OF EXTRINSIC EMULSOME VACCINE CONTAININGSTAPHYLOCOCCUS ENTEROTOXIN B-TOXOID-F COMPLEXED TO PROTEOSOMES

Extrinsic emulsomes formulation of SEB-Toxoid-F-complexed to proteosomeswas performed by preparing plain emulsomes as described in Example 9,but in the absence of the antigen and adding externally the aqueoussolution containing the SEB-Toxoid-F complexed to proteosomes to theplain emulsomes by gently shaking for 15 min at room temperature. Atotal volume of 0.78 ml of stock plain emulsomes were mixed with 0.78 mlsolution of SEB-Toxoid-F (1 mg/ml protein) in 0.15M NaCl and 0.01M Trisbuffer to give a final SEB-Toxoid-F concentration of 0.5 mg/ml.

Example 13 PREPARATION OF INTRINSIC EMULSOME VACCINE CONTAINING LC-467LEISHMANIA LIPOPEPTIDE ANTIGEN

Antigen description and background:

The gene for a surface protein antigen of Leishmania major gp63, hasbeen cloned and sequenced. This protein, recombinantly expressed in liveSalmonella, or given in a sub-unit vaccine as either the purified nativegp63 or selected gp63 peptides (Jardim A., Alexander J., Teh S., Ou D,Olafson R. W. 1990. J. Exp. Med. 172, 645), has recently been shown tolimit the extent of lesion development in murine models of cutaneousleishmaniasis when given with certain adjuvants. These results suggestthat a vaccine to protect humans against leishmaniasis composed ofdefined purified components is a realistic goal. The sub-unit vaccineswere effective, however, only when administered with adjuvantscontaining Corynebacterium parrum (CPV) and poloxamer 407. Otheradjuvants (Complete Freund's Adjuvant, CFA), or lack of adjuvantexacerbated disease. Major success was achieved with the discovery thatsubcutaneous immunization with one small gp63 peptide covalentlyconjugated to lauryl-cysteine protected against severe Leishmaniacutaneous lesions with reduction of lesions in three separateexperiments.

Preparation of emulsomes:

To a round 0.25 liter round-bottomed flask, 0.4 gr of egg-lecithin, 0.4gr of tricaprin, 15 mg of cholesterol, and 15 mg of oleic acid, and 1.5mg of tocopherol succinate were added. The lipid mixture was dissolvedin 50 ml of chloroform. The organic solvent was evaporated untilcomplete dryness under reduced pressure using a rotary evaporator(Heidolph, Germany). To the dry lipid film 50 ml of aqueous solutioncontaining 80 μg of LC-467 Leishmania lipopeptide antigen in phosphatebuffered saline were added and the mixture was then hydrated by shakingfor 30 min. using a multiwrist shaker (Labline, U.S.A.) until all thelipids were homogeneously dispersed in the aqueous phase. The dispersionwas homogenized using a Microlab 70 Gaulin Homogenizer (5 cycles at 800bar). The particle size distribution of the resultant emulsomes wasdetermined using a N4MD Coulter Particle Size Analyzer (CoulterElectronics, England). The differential weight % mode of the instrumentindicated the existence of a single homogeneous population of emulsomeswith a mean particle diameter of 181±35 nm. The emulsome vaccineformulations were then 6.5-fold concentrated using a Filtronultrafiltration stirred cell (Omega Series membrane with 10,000molecular weight cutoff, Filtron Technology Corp., Massachusettes). Theestimated final antigen concentration in the formulation was 0.25 mg/ml.

Example 14 IMMUNOGENICITY OF EMULSOME VACCINE CONTAINING LC-467LEISHMANIA LIPOPEPTIDE ANTIGEN

The objective in the present example was to demonstrate immunogenicityand efficacy of LC-467 Leishmania lipopeptide emulsome vaccine toprotect against severe morbidity of cutaneous leishmaniasis in murinemodels.

The antigen used were lipopeptides obtained from the major glycoproteinof the Leishmania parasite. The peptide denoted 467 was covalentlyattached to lauryl cystsine to serve as the hydrophobic foot.

The murine model used, CBA mouse, resembles the human cutaneous disease.The immunization protocol included two injections of the animals (8mice/group) at weeks 0 and 3 with the experimental vaccines (50 μgpeptide/mouse). At week 6 the mice were infected with live Leishmaniaparasites and the lesion size as function of time was measured andcompared. The results were expressed as % decrease from control (salineinjection). Immunization of the mice with the LC-467 emulsomeformulation enhanced the protective effects (reduction of lesion size)obtained with the free antigen (Table 2).

Since there is considerable homology among Leishmania strains, thispeptide may have wide application in ameliorating lesions caused byother forms of Leishmania.

                  TABLE 2                                                         ______________________________________                                                       % Protection                                                                  (reduction of lesion size                                      Vaccine Formulation                                                                          compared to control mice)                                      ______________________________________                                        LC-467 in saline                                                                             73                                                             LC-467 in emulsomes                                                                          94                                                             ______________________________________                                    

Example 15 ENHANCED MURINE IMMUNOGENICITY OF SEB TOX-F ANTIGEN AFTERPARENTERAL IMMUNIZATION WITH INTRINSIC EMULSOME VACCINE COMPARED TO FREEANTIGEN OR ALUM-ADJUVANTED VACCINE

The antigen used was Staphylococcal Enterotoxin B (SEB) formalinizedtoxoid F. This antigen was formulated intrinsically in emulsome asdescribed in Example 9, and compared to SEB toxoid-F alone or adjuvantedwith alum. BALB/c mice, 5 animals/group, were immunized twice atapproximately 3-week intervals by intramuscular injections with 50 μgdoses of SEB Toxoid F. Sera, obtained after first and secondimmunizations, were analyzed by ELISA techniques using anti-SEB as thedetecting antibody. As shown in FIG. 5, the intrinsic emulsomeformulation was more effective in enhancing immunity to SEB antigens.The anti-SEB serum IgG titers obtained with the emulsome vaccine werehigher than those obtained with the alum-adjuvanted formulation or freeantigen.

Example 16 ENHANCED LAPINE IMMUNOGENICITY OF SEB TOX-F ANTIGEN AFTERPARENTERAL IMMUNIZATION WITH EXTRINSIC EMULSOME VACCINE COMPARED TO FREEANTIGEN

The antigen used was Staphylococcal Enterotoxin B (SEB) formalinizedtoxoid F. This antigen was formulated extrinsically in emulsomes asdescribed in Example 10, and compared to SEB toxoid-F free antigen.Rabbits, 5 animals/group, were immunized twice at approximately 3 weekintervals by intramuscular injections with 100 μg doses of SEB Toxoid F.Sera, obtained after first and second immunizations, were analyzed byELISA techniques using anti-SEB as the detecting antibody. As shown inFIG. 6, the extrinsic emulsome formulation enhanced 4-fold IgG antibodyproduction compared to the free antigen.

Example 17 PROTECTION AGAINST SYSTEMIC CHALLENGE WITH SEB IN MICEIMMUNIZED PARENTERALLY WITH SEB TOXOID VACCINES FORMULATED WITH ALUM,EMULSOME, OR FREE ANTIGEN

Mice immunized parenterally (Table 3) with Staphylococcus Enterotoxin Bin mice immunized with SEB Toxoid-F vaccine formulated with emulsomes(as described in example 9) were significantly protected againstsystemic SEB challenge (100 ug toxin).

                  TABLE 3                                                         ______________________________________                                                  Formula- Anti-SEB    Died/                                          Antigen   tion     IgG         total                                                                              Survival                                  ______________________________________                                        control   --       0            9/10                                                                              10%                                       SEB-tox F --       3,200       3/5  40%                                       SEB-tox F Alum     51,200      4/5  20%                                       SEB-tox F Emulsome 102,400     0/5  100%                                      ______________________________________                                    

The data in Table 3 show a very good correlation between the anti-SEBserum IgG titers obtained after intramuscular immunization of CD-1 micewith protection against systemic challenge with 100 g of SEB. In thegroups immunized with intrinsic emulsome-SEB Toxoid F vaccine, thesurvival was 100% while for animals immunized with free antigen oralum-adjuvanted vaccine the survival was only 20 to 40%.

Example 18 PROTECTION OF MICE IMMUNIZED WITH MUCOADHESIVE EMULSOMEVACCINES CONTAINING SEB-TOXOID F ANTIGEN OR SEB-TOX F COMPLEXED TOPROTEOSOMES AGAINST INTRANASAL CHALLENGE WITH SEB TOXIN IN BALB/C MICED-GALACTOSAMINE MODEL

BALB/C mice (10 animals/group) were immunized intranasally twice with100 μg antigen doses of Mucoadhesive Emulsome vaccines containing eitherSEB-Tox F antigen or SEB-Tox F complexed to proteosomes. Themucoadhesive formulations were prepared as described in Example 4. Theantigens were added extrinsically to the prepared plain emulsomes. Aftera second immunization mice were challenged intranasally with an LD100dose (350 μg) of SEB Toxin. Mice survival was determined 3 dayspost-challenge. FIGS. 7A and 7B clearly show that the mice immunizedwith the emulsome vaccines had the highest percent of survival either inthe Tox F or Tox F complexed to proteosomes groups (70% and 100%survival, respectively,compared to 0 survival in the control group).

Example 19 INDUCTION OF MUCOSAL, INTESTINAL, AND SYSTEMIC IMMUNOGENICITYIN MICE VACCINATED INTRANASALLY WITH ANTI-HIV ENVELOPE gp160 ANTIGENALONE OR COMPLEXED TO PROTEOSOMES INCORPORATED IN EXTRINSIC MUCOADHESIVEEMULSOME VACCINES

The antigens used were gp160 alone or gp160 complexed to meningococcalouter membrane proteosomes. These antigens were formulated extrinsicallyin emulsomes as described in Example 8. Mice (5 animals/group) wereimmunized intranasally with 100 μg antigen doses at different intervals.Murine sera, lung fluids, and intestinal fluids obtained afterimmunizations were analyzed by ELISA techniques using several specificHIV epitopes as the detecting antigens.

Table 4 shows that emulsomes enhanced the mucosal immunogenicity of HIVenvelope protein following intranasal immunization with HIV gp160formulated with or without the proteosome vaccine delivery system.Specific systemic anti-HIV IgG antibody production was enhanced 8-foldin the sera of mice immunized with the antigen incorporated in emulsomevaccine formulation, and 128-fold in the sera of mice immunized with thegp160-proteosome-emulsome formulation compared to the free antigen. Inaddition, immunization with gp160 plus emulsomes without proteosomesenhanced anti-gp160 serum IgA titers of 25,600 compared to <50 obtainedby immunizing with the free antigen as well as 270-fold and 6-foldincreases in anti-gp160 intestinal and lung fluid IgA, respectively,compared to immunizing with gp160 alone. Table 4 also shows thatintranasal immunization with HIV gp160 formulated with emulsomesenhances serum IgG HIV gp41 peptide epitope responses (C448, C41, andCKen) as measured by quantitative western blot analyses. In addition,formulation of the gp160-proteosome vaccine with emulsomes enhances theserum IgG HIV responses to each of seven gp120 and gp41 peptide epitopestested with increased responses ranging from over 116,000 to 9,000,000.

These data show that optimal antigen delivery to the mucosal immunesystem is effected by formulating with emulsomes and that specificsecretory IgA antibodies can be elicited and boosted even far away fromthe immunizing site. Lung and intestinal immunity was improved afternasal immunization.

                  TABLE 4*                                                        ______________________________________                                        Vaccine                                                                       Formulation                                                                   ______________________________________                                        Anti-gp160 IgG or IgA titers of sera, gavage, and                             lavage fluids as measured by ELISA:                                           Anti-gp160                                                                              51,200   409,600   1,638,400                                                                             6,553,600                                Serum IgG                                                                     Anti-gp160                                                                              <50      <50       3200    25,600                                   Serum IgA                                                                     Anti-gp160                                                                              5        147       32      1351                                     Intestinal                                                                    IgA                                                                           Anti-gp160                                                                              512      111       1176    3105                                     Lung IgA                                                                      ______________________________________                                        Anti-gp160 peptide igG responses measured by                                  quantitative western blots:                                                   Peptide                                                                       epitope                                                                       Specificity:                                                                  ______________________________________                                        C1 (48-128)                                                                             <100     <100      39,918  987,775                                  C21e (254-                                                                              <100     <100      7354    1,044,098                                274)                                                                          V3 (290-338)                                                                            <100     <100      49,963  116,888                                  C3 (342-405)                                                                            <100     <100      <100    319,355                                  C448 (453-                                                                              <100     182,362   <100    9,025,282                                518)                                                                          C41 (579-605)                                                                           210,167  1,833,409 1,189,986                                                                             2,469,425                                CKen (735-                                                                              <100     73,621    15,119  2,697,628                                752)                                                                          ______________________________________                                         *Bolded numbers indicate enhancement by emulsomes compared to same vaccin     in saline without emulsomes. Underlined numbers indicate enhancement by       proteosomes compared to same formulation without proteosomes.            

Example 20 INCREASED IMMUNE RESPONSE IN RHESUS MONKEYS IMMUNIZED WITHANTI-LEISHMANIA INTRINSIC EMULSOME VACCINE CONTAINING LC-467 LIPOPEPTIDEANTIGEN

Rhesus monkeys (Macacca mulatta, 5 animals/group) were immunizedintramuscularly three times at weeks 0, 4 and 10 with 500 μg doses ofLC-467 Leishmania lipopeptide antigen incorporated intrinsically inemulsomes as described in Example 13. Monkey sera was collected beforeand after each immunization and analyzed for anti-Leishmania IgGantibody levels by ELISA.

The results in FIG. 8 show that the monkey group vaccinated with theemulsome vaccine resulted in the highest anti-Leishmania IgG antibodytiter, whereas the control group and monkeys that received the LC-467antigen alone had very low antibody levels.

Example 21 LYOPHILIZATION OF EMULSOMES

To a 0.5 liter round-bottomed flask, 3.5 g of egg-lecithin, 5.2 g oftricaprin, 0.14 g of cholesterol, 0.14 g of cholesterol, 0.14 g of oleicacid, and 0.05 g of tocopherol succinate were added. The lipid mixturewas dissolved in 50 ml dichloromethane. The organic solvent wasevaporated to complete dryness under reduced pressure using a rotaryevaporator (Heidolph, Germany). To the dry lipid film, 63 ml of a 7%sucrose solution were added, and the mixture was then hydrated byshaking until all the lipids were homogeneously dispersed in the aqueousphase. The dispersion was homogenized for 2 minutes at 13,000 rpm usinga Polytron PT 3000 (Kinematica, AG). The preparation was then submittedto 7 cycles of high shear homogenization at 800 bar using a Microlab70Gaulin Homogenizer. The formulation was divided in 5 ml portions and thevials were then freeze-dried using a Christ Beta Freeze Dryer (Germany).The samples were reconstituted with water for injection and the particlesize distribution of the resultant reconstituted emulsome formulationwas measured. An average size of 102±15 was measured, very similar tothe mean size obtained before lyophilization.

Example 22 PREPARATION OF POLYMERIC EMULSOMES FOR CONTROLLED RELEASE OFANTIGENS

To a 0.5 liter round-bottomed flask, 0.5 g of polylactide ("Resomer L104," MW=2,000 Da, Boehringer Ingelheim, Germany), 0.5 g ofegg-lecithin, 0.5 g of tricaprin, 0.2 g of cholesterol, 0.2 g of oleicacid, and 0.02 g of tocopherol succinate were added. The lipid mixturewas dissolved in 50 ml dichloromethane. The organic solvent wasevaporated until complete dryness under reduced pressure using a rotaryevaporator (Heidolph, Germany). To the dry lipid 50 ml of saline wereadded and the mixture was then hydrated by shaking until all the lipidswere homogeneously dispersed in the aqueous phase. The dispersion washomogenized for 5 minutes at 17,000 rpm using a Polytron PT3000(Kinematica, AG). The preparation was then submitted to 10 cycles ofhigh shear homogenization at 800 bar using a Microlab70 GaulinHomogenizer. To this emulsome formulation, antigens can be addedextrinsically or intrinsically.

The invention has been described with respect to certain specificembodiments thereof. However, those skilled in the art will readilyconceive of many other means of carrying out the present invention basedupon the generic teachings hereinabove. Applicants propose to be bound,therefore, only by the spirit and scope of the invention as reflected inthe appended claims.

What is claimed is:
 1. A pharmaceutical composition for theadministration of antigen which comprises a nanoemulsion of a pluralityof noncellular lipid particles having a mean diameter of about 10 to 250nm, as determined on a weight basis, the particles being suspended in anaqueous continuous phase,wherein each said lipid particle comprises alipid core composed of a lipid which is a solid or liquid crystal asdetermined in bulk at a temperature of about 25° C. or higher, and atleast one phospholipid bilayer surrounding said core and encapsulating aportion of said aqueous continuous phase, said particles entrappingabout 0.001 to 5% of an immunogen in said lipid core or in saidencapsulated aqueous phase.
 2. The pharmaceutical composition of claim 1wherein the mean particle diameter of said lipid particles falls withinthe range of about 20 to 180 nm as determined on a weight basis.
 3. Thepharmaceutical composition of claim 2 wherein the particle diameter ofat least 99% of said lipid particles falls within the range of about 50to 150 nm as determined on a weight basis.
 4. The pharmaceuticalcomposition of claim 2 wherein the lipid core comprises a fatty acidester.
 5. The pharmaceutical composition of claim 4 wherein the lipidcore has a solid to fluid phase transition temperature below 37° C. asdetermined in bulk.
 6. The pharmaceutical composition of claim 4 whereinthe lipid core comprises a triglyceride.
 7. The pharmaceuticalcomposition of claim 6 wherein said triglyceride comprises a fatty acidmoiety of C₁₀ to C₁₈.
 8. The pharmaceutical composition of claim 6wherein said triglyceride is completely saturated.
 9. The pharmaceuticalcomposition of claim 6 wherein said triglyceride is selected from thegroup consisting of tricaprin, trilaurin, trimyristin, tripalmitin, andtristearin.
 10. The pharmaceutical composition of claim 6 wherein themole ratio of phospholipid to total lipid is in the range of from 0.1:1to 0.5:1.
 11. The pharmaceutical composition of claim 6 wherein theweight ratio of phospholipid to triglyceride is in the range of from0.5:1 to 1.5:1.
 12. The pharmaceutical composition of claim 4 whereinsaid phospholipid is a phosphatidylcholine.
 13. The pharmaceuticalcomposition of claim 12 wherein said phosphatidylcholine is eggphosphatidylcholine.
 14. The pharmaceutical composition of claim 12wherein said phosphatidylcholine has a transition temperature below 25°C.
 15. The pharmaceutical composition of claim 12 wherein saidphosphatidylcholine is saturated.
 16. The pharmaceutical composition ofclaim 1 wherein said lipid particle contains cholesterol or cholesterylesters.
 17. The composition of claim 1 wherein the immunogen ishydrophilic, lipophilic, or amphophilic.
 18. The composition of claim 1wherein the immunogen is a peptide, protein, or glycoprotein.
 19. Thecomposition of claim 18 wherein the antigen is the gp160 envelopeprotein of the HIV virus, or a fragment thereof.
 20. The composition ofclaim 18 wherein the antigen is the surface glycoprotein of a Leishmaniaparasite, or a fragment thereof.
 21. The composition of claim 20 whereinthe surface glycoprotein or peptide is covalently conjugated to ahydrophobic component.
 22. The composition of claim 21 wherein thehydrophobic component is lauryl-cysteine.
 23. The composition of claim 1wherein the immunogen is a protein toxoid.
 24. The composition of claim23 wherein the immunogen is Staphylococcus Enterotoxin B toxoid.
 25. Thecomposition of claim 1 wherein the immunogen is complexed with aproteosome.
 26. The composition of claim 1 wherein the nanoemulsionfurther comprises a bioadhesive or mucoadhesive macromolecule.
 27. Thecomposition of claim 26 wherein the said mucoadhesive macromolecule is apolymer.
 28. The composition of claim 26 wherein the said mucoadhesivemacromolecule is selected from the group of acidic nonnatural polymersconsisting of polymers and copolymers containing acrylic acid units,polymers and copolymers containing methacrylic acid units, andpoly(methylvinylether/maleic anhydride) copolymer.
 29. The compositionof claim 28 wherein the said polymer is polyacrylic acid.
 30. Thecomposition of claim 1 which contains no added muramyl peptides.
 31. Thepharmaceutical composition of claim 1 wherein said lipid particle issubstantially free of lipase and phospholipase activity.
 32. A methodfor delivery of an immunogen to an animal, comprising administering tosaid animal a pharmaceutical according to claim
 1. 33. The method ofclaim 32 wherein the mean diameter of the lipid particles in saidcomposition is in the range of about 20 to 180 nm.
 34. The method ofclaim 32 wherein said composition is administered parenterally, orally,intranasally, or topically, thereby providing enhanced immunogenicity.35. The method of claim 32 wherein said composition is administered tomucosal surfaces, thereby achieving mucosal immunity.
 36. A method formaking a nanoemulsion for administration of an immunogen, comprising thesteps of:preparing a mixture comprising phospholipid and triglyceride inthe weight ratio range of about 0.5:1 to 1.5:1 wherein said triglyceridehas a solid to liquid phase transition temperature of greater than 25°C.; suspending said mixture in an aqueous solution at a temperaturebelow the solid to liquid transition temperature of the triglyceride;reducing the size of the suspension to yield a nanoemulsion of lipidparticles having a mean particle diameter of between about 10 nm and 250nm; and incorporating an immunogen in the nanoemulsion.
 37. The methodaccording to claim 36 for preparing the composition of the nanoemulsionby an intrinsic procedure, where the immunogen is added beforehomogenization of water and oil phases.
 38. The method of claim 36 forpreparing the composition of the nanoemulsion by an extrinsic procedure,where the immunogen is added externally by mixing with the previouslyprepared plain nanoemulsion.
 39. A pharmaceutical composition comprisingdehydrated lipid particles containing an antigen for administration as ananoemulsion, wherein said lipid particles comprise a lipid coresurrounded by at least one phospholipid layer, said lipid core iscomposed of lipid in a solid or liquid crystalline phase at least about25° C. as determined in bulk, and said lipid particles have a meandiameter upon rehydration of about 10 to 250 nm.
 40. The pharmaceuticalcomposition of claim 39 further comprising a cryoprotectant.
 41. Thepharmaceutical composition of claim 40 wherein said cryoprotectant isselected from the group consisting of glucose, sucrose, lactose,maltose, trehalose, dextran, dextrin, cyclodextrin,polyvinylpyrrolidone, and amino acids.
 42. The pharmaceuticalcomposition of claim 40 wherein said cryoprotectant is present in arange of from 0.1% to 10% by weight compared to lipid.
 43. Thepharmaceutical composition of claim 39 wherein said lipid particlescontain an immunogen.
 44. A method for delivering an antigen to ananimal comprising administering to said animal a pharmaceuticalcomposition according to claim 39.